Oligonucleotides which are partially complementary to genomic DNA sequence can associate with duplex DNA in a sequence specific manner. See, e.g., WO2011/133802. Oligonucleotides comprising sequences that vary in part from corresponding genomic DNA sequences have been used to edit chromosomal DNA by recombination of the sequence encoded by the oligonucleotide into the genome.
Whereas oligonucleotides are also used in biological research, a main intended use of oligonucleotides in cell therapy is to correct genetic sequences associated with disease in cells to produce preparations of therapeutic cells for use. However, the efficiency of oligonucleotide-mediated alteration of the genome is very low, limiting the applicability of this approach. At the same time, due to the inefficiency of the process, it is thought that off-target alteration of the genome is also low. Consequently, methods that utilize oligonucleotides to produce therapeutic cells without increasing their low expected off-target activity and that produce increased yields of optimally-modified cells would be of interest to enable more effective and safe cell therapy.
The sequence of an oligonucleotide used to introduce a genetic alteration is typically designed to correspond to one strand of a target double stranded chromosomal DNA sequence of interest, usually the coding sequence or promoter of a gene of interest wherein the oligonucleotide is additionally designed to comprise a mismatch compared to the target sequence. Oligonucleotides are thought to associate with their corresponding target sequences in the genome. In some cases, the mismatched sequence is introduced into the genome following recombination events.
The efficiency of oligonucleotide-mediated alteration of the genome is low. Various publications teach that oligonucleotides should be designed to contain sequences that are essentially identical to their corresponding target sequences in order to work. For example, US 2010/0172882 teaches that a greater number of homologous positions within the oligonucleotide will increase the probability that it will be recombined into the target sequence, target region or target site. Thus, having more mismatches is discouraged by existing publications. Many oligonucleotides used to alter the genome via recombination reported to date have been designed to introduce a point mutation or to change a single base. See, e.g., Bonner M., et al. 1:e18. doi: 10.1038/mtna.2012.9 Mol. Ther. Nucleic Acids (2012); Bertoni C, et al. 37(22):7468-82. doi: 10.1093/nar/gkp757 Nucleic Acids Res. (2009) and Andrieu-Soler C, et al. Nucleic Acids Res. 7; 33(12):3733-42 (2005).
In order to increase the efficiency of genome editing using oligonucleotides, other references (e.g., US 2011/0262406) teach the use of triplex-forming molecules, which is described as a pair of single-stranded molecules, or a single molecule composed of a pair of molecules connected by a linker, that facilitate strand displacement and triplex formation, in which one molecule binding to the target strand by Hoogsteen binding and the other molecule binds to the target strands by Watson-Crick binding in a sequence specific manner. It is thought that these molecules recruit cellular factors that are involved in recombination which work to increase the efficiency with which oligonucleotides designed to edit the genome are recombined into the genome, and they have been used in editing the genome of cells for the treatment of various diseases, such as HIV. The use of multiple compounds here is one way of approaching genomic editing. Nonetheless, even in combination with these molecules, the efficiency of genome modification using oligonucleotides remains low. There is a need to solve the problem of inefficient recombination, inefficient genomic editing, and increased off-target activity, as well as additional benefits of providing for compositions and methods of producing therapeutic cells without increasing their low expected off-target activity and increasing yields of optimally-modified cells.
Human immunodeficiency virus (HIV) has infected millions of people worldwide. Many efforts have been made to combat HIV infection. Some approaches focus on small molecules to affect the virus' replication cycle. Other efforts have focused on the cell therapy side. Since CCR5 is known to be a co-receptor that is needed for many HIV isolates, efforts have been made to target CCR5 for HW therapy. See, e.g., WO 2009/06336, US 2005/0220772, and US 2009/0202496.
The reported cure of the Berlin Patient has spurred efforts to create cell therapies based on the interpretation that any disruption of CCR5 may underlie a safe and effective cell therapy. One report on a patient study referred to as the “Berlin Patient” described how the patient was cured following a bone marrow transplant using bone marrow derived from a 432 homozygous individual, or an individual carrying only this particular variant of the gene and no wild-type or other variant. See, e.g., Allers K, et al. Blood 117(10): 2791-9 (2011). The 432 variant of CCR5 represents a disrupted variant of the wild-type gene which occurs in a small percentage of North Americans and Europeans but is almost nonexistent in Asians and Africans. In order to infect its target cells, HIV must first dock onto its receptor and co-receptor, CD4 and CCR5, respectively, both of which are expressed on the surface of target cells. Δ32 homozygous individuals are naturally-resistant to HIV/AIDS and otherwise healthy. Thus, it was thought that disruption of CCR5 per se may be used as the basis of an HIV/AIDS cell therapy. However, various mutations of CCR5 (e.g. introduction of stop codon(s)) have not proven to be viable cell therapy approaches.
Attempts have been made to isolate different types of cells, such as stem cells (e.g., cord blood-derived stem cells and hematopoietic stem cells) that have naturally-occurring Δ32 mutations and to expand these isolated stem cells with naturally-occurring Δ32 mutation. However, there has been not much success with expansion of populations of stem cells with naturally occurring Δ32 mutations. There exists many problems with making the appropriate mutations in CCR5 that render the modified cell to be less likely to be infected with HIV. As such, one problem to be solved is the modification of CCR5 in cells that are susceptible to HW infection such that the resultant cells are less likely to be infected with HIV. In addition, another problem is the isolation and/or expansion of stem cells with the precise Δ32 mutations to the extent that there are enough cells for cell therapy for individual infected with HIV, suspected of having HIV infection or at risk of HIV infection. Furthermore, another problem exists with isolating and/or purifying the right population of stem cells with the precise Δ32 deletion in CCR5.